Nitrous oxide has been used as a powerful analgesic for centuries, but little is known of its cellular mechanism of action. This proposal outlines our plans to explore the mechanism of nitrous oxide's effect on ion channels, which has not yet been described. Nitrous oxide (N2O) is of particular interest because it is one of the few agents that are selective for the CaV3.2 isoform of T-type calcium channels. Interestingly, this particular channel has been recently implicated in the pain pathway (Choi et al., 2006). Thus, we hope to describe the mechanism of N2O's effect on T-channels and at the synapse. We also plan to explain the relationships between N2O, the pain pathway, and T-type via behavioral studies of CaV3.2-null mice. Pharmacological and electrophysiological studies in dorsal root ganglion neurons are planned to continue our preliminary studies of N2O's mechanism of action. Previous studies and our recent data suggest that N2O block of CaV3.2 channels is dependent on the presence of trace metals, one extracellular histidine that is capable of binding metals, and free oxygen radicals that have the ability to modify metals and proteins. Based on this information, we plan to further explore the details of these interactions. Secondly, we propose an investigation into the effects of N2O on synaptic transmission in dorsal root ganglia-dorsal horn (DRG-DH) co-cultures. This first synapse in the pain pathway has been shown to be the site of central sensitization and the target of some general anesthetics. N2O inhibits currents through NMDA receptors and T-channels, which are both present at this synapse. Thus we hypothesize that N2O may exert some of its analgesic effects here. Lastly, the proposed behavioral pain testing on CaV3.2 knockout mice after exposure to N2O will shed light on the role of T-type channels in mediating N20 analgesia. We plan to test multiple types of pain, including chemical, mechanical, and thermal pain for a comprehensive study. If CaV3.2 channels mediate a portion of N2O analgesia, we expect that these knockout mice will display decreased sensitivity to the analgesic effects of N2O. This proposal aims to describe the unexplained molecular mechanism of a commonly used clinical analgesic, N2O. We also plan to explore N20's action on calcium channels that have already been strongly implicated in the modulation of pain. Greater knowledge of the role that these channels play in pain processing and N2O-induced analgesia will advance the treatment of chronic and neuropathic pain, and provide a better understanding of currently used anesthetics.